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Published on: February 13, 2019
Cardiac expression and location of hexokinase changes in a mouse model of pure creatine deficiency
Jelena Branovets1, Niina Karro1, Karina Barsunova1
1Laboratory of Systems Biology, Institute of Cybernetics, Tallinn University of Technology, Tallinn, Estonia.
Insights
In creatine-deficient mice, heart energy transfer systems like hexokinase (HK) and adenylate kinase (AK) do not fully compensate for limited creatine kinase (CK). AGAT knockout mice showed altered HK, impacting mitochondrial function.
Area of Science:
- Cardiovascular Physiology
- Mitochondrial Metabolism
- Enzymology
Background:
- Creatine kinase (CK) is the primary phosphotransfer system in the heart, crucial for energy buffering and mitochondrial respiration.
- Creatine deficiency, induced by AGAT or GAMT gene knockout, limits CK function, necessitating investigation into alternative energy transfer systems.
- Hexokinase (HK) and adenylate kinase (AK) are potential alternative pathways for ADP channeling and energy transfer in cardiomyocytes.
Purpose of the Study:
- To investigate the expression, activity, and mitochondrial coupling of HK and AK in creatine-deficient mouse hearts.
- To determine if HK and AK can compensate for the substrate-limited CK system in AGAT and GAMT knockout mice.
- To assess the impact of altered phosphotransfer systems on mitochondrial respiration and function.
Main Methods:
- Assessment of mitochondrial respiration stimulated by endogenous ADP generated by HK, AK, and CK in permeabilized cardiomyocytes.
- Measurement of AK, CK, and HK activities in whole heart homogenates and subcellular fractions (cytosolic and mitochondrial).
- Evaluation of the expression levels of major HK, AK, and CK isoforms in wild-type and knockout mouse hearts.
Main Results:
- In AGAT knockout (KO) mice, a model of pure creatine deficiency, HK activity increased in the mitochondrial fraction, and HK I expression was greater, but HK-stimulated respiration decreased.
- Adenylate kinase (AK) activity and expression remained unchanged in both AGAT KO and GAMT KO mice, indicating it does not compensate for CK deficiency.
- While CK-stimulated respiration was high (80%), HK and AK contributed less (25% and 90% respectively), with limited mitochondrial channeling for HK (25%) and none for AK.
- GAMT KO mice showed no significant changes in AK, CK, or HK activity, distribution, or expression compared to controls.
Conclusions:
- The mouse heart relies less on phosphotransfer systems for ADP flux across the mitochondrial membrane than previously thought.
- In pure creatine deficiency (AGAT KO mice), altered HK expression and activity may influence mitochondrial regulation and reactive oxygen species production.
- Adenylate kinase does not appear to be a significant compensatory phosphotransfer system for creatine kinase in the heart under these conditions.
Abstract:
Creatine kinase (CK) is considered the main phosphotransfer system in the heart, important for overcoming diffusion restrictions and regulating mitochondrial respiration. It is substrate limited in creatine-deficient mice lacking l-arginine:glycine amidinotransferase (AGAT) or guanidinoacetate N-methyltranferase (GAMT). Our aim was to determine the expression, activity, and mitochondrial coupling of hexokinase (HK) and adenylate kinase (AK), as these represent alternative energy transfer systems. In permeabilized cardiomyocytes, we assessed how much endogenous ADP generated by HK, AK, or CK stimulated mitochondrial respiration and how much was channeled to mitochondria. In whole heart homogenates, and cytosolic and mitochondrial fractions, we measured the activities of AK, CK, and HK. Lastly, we assessed the expression of the major HK, AK, and CK isoforms. Overall, respiration stimulated by HK, AK, and CK was ∼25, 90, and 80%, respectively, of the maximal respiration rate, and ∼20, 0, and 25%, respectively, was channeled to the mitochondria. The activity, distribution, and expression of HK, AK, and CK did not change in GAMT knockout (KO) mice. In AGAT KO mice, we found no changes in AK, but we found a higher HK activity in the mitochondrial fraction, greater expression of HK I, but a lower stimulation of respiration by HK. Our findings suggest that mouse hearts depend less on phosphotransfer systems to facilitate ADP flux across the mitochondrial membrane. In AGAT KO mice, which are a model of pure creatine deficiency, the changes in HK may reflect changes in metabolism as well as influence mitochondrial regulation and reactive oxygen species production.NEW & NOTEWORTHY In creatine-deficient AGAT-/- and GAMT-/- mice, the myocardial creatine kinase system is substrate limited. It is unknown whether subcellular localization and mitochondrial ADP channeling by hexokinase and adenylate kinase may compensate as alternative phosphotransfer systems. Our results show no changes in adenylate kinase, which is the main alternative to creatine kinase in heart. However, we found increased expression and activity of hexokinase I in AGAT-/- cardiomyocytes. This could affect mitochondrial regulation and reactive oxygen species production.
